Method of inhibiting replication of hyperproliferative cells

ABSTRACT

This invention relates to methods for converting a pathologic hyperproliferative human cell to its non-malignant phenotype. The method comprises introducing into the cell a nucleic acid encoding a polypeptide having adenovirus E1A activity and growing the cell under conditions such that the polypeptide is produced. This invention also relates to a method of converting a population of pathologic hyperproliferative cells in a subject to a non-hyperproliferative state by expressing, in some but not all of the hyperproliferative cells, an isolated nucleic acid sequence encoding a polypeptide having adenovirus E1A activity. In a further aspect, the invention relates to promoting differentiation of pathologically hyperproliferative cells.

The present invention was made with support in part from a National Institutes of Health Grant 7-R29-GM-44573-03 and an Institutional Support Grant Account No. 1-17071-6460. The United States Government may have certain rights in the invention.

BACKGROUND OF THE INVENTION

Adenovirus is a large DNA virus whose natural host is human cells. Virtually every adult has been infected with adenovirus at some time, the major effect being cold-like symptoms. Adenovirus is referred to as a "DNA tumor virus" purely because of its oncogenic effect in rodents. Expression of the adenovirus genome occurs in two stages. First, the early gene products are expressed which encode the E1A and E1B genes. These products are necessary for expression of the late gene products. Late gene products encode proteins necessary for replication as well as the viral structural proteins.

The proteins encoded by the E1A gene of adenovirus have been studied primarily from two points of view. First, the 243 amino acid and 289 amino acid forms of E1A (arising from alternative splicing of the precursor RNA such that the 243 amino acid protein is a subset of the 289 amino acid protein) are both transcriptional regulatory proteins, J. Flint, T. Shenk, Ann. Rev. Gen. 23:141-161 (1989). Secondly, these proteins facilitate the oncogenic transformation of certain rodent cells by other oncogenes, H. E. Ruley, Nature 304:602-606 (1983), and, as such E1A is generally classified as an oncogene. The interactions of E1A proteins with various defined cellular proteins are believed to mediate its oncogenic effects in rodent cells, J. M. Howe, et al., Proc. Natl. Acad. Sci. 87:5883-5887 (1990).

In human cells, E1A does not appear to cooperate with other oncogenes to transform cell lines, however, mild tumorigenic effects have been reported in only two instances. For example, embryonic kidney, F. Graham, J. Smiley, W. Russell, R. Nairn, J. Gen. Virol. 36:59-72 (1977), and embryonic retinoblast cell lines, P. J. Byrd, K. W. Brown, P. H. Gallimore, Nature 298:67-71 (1982), stably expressing E1A are weakly tumorigenic when injected into mice. Moreover, these transformants arise very rarely after cotransfection of E1A with another oncogene.

The normal and transformed phenotypes of a cell are thought to be maintained by a balance of activities encoded by oncogenes and antioncogenes (tumor suppressor genes). The absence of functional tumor suppressor gene products contributes to carcinogenesis. Conversely, replacement or overexpression of these products promises to be useful in controlling tumor cell growth. Only a limited number of tumor suppressor genes have been characterized at present, notably, retinoblastoma protein, Huang et al., Science 24:1563-1566 (1988), and p53, Chen et al., Science 250:1566-1570 (1990). However, when reintroduced into tumor cells, both of these gene products simply kill the cells.

Two reports in the literature claim tumor suppression effects of E1A, but both of these effects arose from epiphenomena not related to the growth controlling effects of E1A expression. Rat sarcoma cells stably transfected with E1A are less tumorigenic in nude rats than their untransfected counterparts (T. A. Walker, B. A. Wilson, A. M. Lewis, J. L. Cook, Proc. Natl. Acad. Sci. 88:6491-6495 (1991). Decreased tumorigenicity of E1A-expressing cells resulted from sensitization of these cells to cytolysis by natural killer cells and these cells were still tumorigenic in nude mice. The second report (D. Yu, K. Scorsone, M. C. Huang Molec. Cell Biol. 11:1745-1750 (1991) showed that E1A prevented an oncogene known as neu from transforming the mouse cell line 3T3. E1A accomplished this solely by inhibiting the expression of neu at the transcriptional level. 3T3 cells transfected with a chimeric neu gene that utilized an E1A-resistant gene promoter were still transformed by neu even in the presence of E1A. The use of E1A to suppress tumor proliferation in the manner implied by the observations above would require E1A expression in all of the cells in the tumor. This requirement poses extreme practical limitations on the use of tumor suppressor genes for therapy since the technology does not exist to introduce a desired gene into 100% of the cell population. In light of these drawbacks, strategies for inhibiting the uncontrolled replication of hyperproliferative cells using tumor suppressor genes have not yet emerged.

Thus, there exists a need to regulate hyperproliferative cell populations through small subpopulations of cells endowed with the ability to dominantly influence their growth. The present invention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

This invention relates to methods for converting a pathologic hyperproliferative human cell to its non-malignant phenotype. The method comprises introducing into the cell a nucleic acid encoding a polypeptide having adenovirus E1A activity and growing the cell under conditions such that the polypeptide is produced. This invention also relates to a method of inhibiting the growth of a population of pathologic hyperproliferative cells in a subject by expressing, in some but not all of the hyperproliferative cells, an isolated nucleic acid sequence encoding a polypeptide having adenovirus E1A activity. In a further aspect, the invention relates to promoting differentiation of pathologically hyperproliferative cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Demonstration that E1A can be stably expressed in transfected tumor cells. Immunoprecipitation of E1A proteins from stably transfected and parental cell lines. [35S]Methionine-labeled proteins from E1A -expressing clones derived from HT1080 cells (lanes 1-5), HeLa cells (lanes 6-8), or A2058 cells (lanes 9-11) were immunoprecipitated with anti-E1A antibodies (E) or control antibodies (C). Lanes: 1-5, HT1080, p2AHT2a, p1Aneo3, p1Aneo15, and p1Aneo16, respectively: 6-8, HeLa, Medg18, and Medg28, respectively: 9-11, A2058, 1A58c8-1, and 1A58c11-1, respectively. The bands corresponding to E1A protein species are bracketed.

FIG. 2. Demonstration that E1A expressing cells are converted into a flat, contact-inhibited morphology. Phase-contrast micrographs of parental (top row), neomycin-resistance transfected (middle row), and E1A-expressing (bottom row) human tumor cells. Bottom row, photomicrographs of HT1080neo^(r) and E1A-expressing HT1080 soft agarose colonies.

FIG. 3. Demonstration that E1A expression causes loss of anchorage-independent growth (soft agarose colony formation) in transfected tumor cells. Soft agarose colony formation assays of parental neomycin-resistance transfected, and E1A-expressing human tumor cells.

FIG. 4. Demonstration that E1A reduces tumorigenicity of human tumor cells. Tumorigenicity of parental tumor cells (HT1080, A2058, and HeLa) and E1A derivatives in nude mice.

FIG. 5. DNA sequence of E1A coding sequences (cDNA) (SEQ. ID. NOS. 1-4). Bottom line: 243 amino acid sequence (SEQ. ID. NOS. 1 and 2). Top line: 289 amino acid sequence (SEQ. ID. NOS. 3 and 4).

FIG. 6. Structure of a reverse-transforming retrovirus construct. The 243 amino acid form of E1A (12S cDNA) was cut at adenovirus map position 610 with BstX1 and the 5' end was reconstructed with a double-stranded oligonucleotide, thus removing all E1A 5' noncoding sequences. The HindIII end of this oligonucleotide was made blunt ended and the 3' end of E1A was cut with HpaI (map position 1575) to remove the polyA addition site. The resulting E1A sequence was subcloned into the retrovirus vector LNSX (described in Miller and Rosman, Biotechniques 7:980-990 (1989). Retroviruses were then packaged as described in the text.

DETAILED DESCRIPTION OF THE INVENTION

The present invention results from the observation that the adenovirus E1A gene unexpectedly influences the phenotype of human tumor cells so as to restore their contact inhibitory properties and promote their differentiation. Stable expression of the adenovirus 5 E1A gene reduces anchorage-independent growth and tumorigenic potential, promotes reorganization, induces flat morphology, and restores contact inhibition in human tumor cell lines. By these criteria, E1A functions as a tumor suppressor gene in this human context. The apparent paradox accorded by the observations of the ability of E1A to transform rodent cells in cooperation with other oncogenes suggests that E1A may be the prototype of a class of growth-regulatory proteins having context-specific transforming and antioncogenic activities.

These unexpected observations provide the basis for this invention, which in one embodiment provides a method for reverse-transforming pathologic hyperproliferative mammalian cells. The cells are contacted with an effective amount of nucleic acid encoding a polypeptide having E1A activity under conditions such that the nucleic acid is introduced into the cell. The cell is then grown under conditions such that the polypeptide is expressed. The method may be practiced in vitro or in vivo.

Provided by this invention is a method for reversing the transformed phenotype of hyperproliferative cells located in pathological tissue by transducing with, for example, retroviral vectors, isolated nucleic acids having E1A activity. Transducing vectors can be introduced by direct injection or other suitable application. Such reverse-transformed cells will arrest growth of neighboring, non-transduced cells.

Alternatively, provided by this invention is a method of reverse-transforming a population of pathologic hyperproliferative cells in a subject which comprises obtaining a suitable sample of cells from the subject, contacting the cells with a suitable retroviral vector containing nucleic acid encoding a polypeptide having E1A activity for a time period for transfection of the nucleic acid into the cell. These cells are then grown under suitable conditions such that the nucleic acid is expressed. The actively expressing cells are then reinfused into the subject.

As used herein, the term "hyperproliferative," as it relates to cells, includes but is not limited to those cells having the capacity for autonomous growth, i.e., existing and reproducing independently of normal regulatory mechanisms. Hyperproliferative diseases may be categorized as pathogenic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state. "Pathologic hyperproliferative" cells occur in disease states characterized by malignant tumor growth including but not limited to breast cancer, sarcomas and other neoplasms, bladder cancer, colon cancer, lung cancer, various leukemias and lymphomas. In addition, the term can be applied to such conditions as thyroid hyperplasia and benign prostatic hypertrophy. Examples of non-pathologic hyperproliferative cells are found, for example, in embryonic tissue, in mammary ductal epithelial cells during development of lactation and also in cells associated with wound repair. Pathologic hyperproliferative cells characteristically exhibit loss of contact inhibition and a decline in their ability to selectively adhere which implies a change in the surface properties of the cell and a further breakdown in intercellular communication.

The sequence of E1A is provided in FIG. 5 (SEQ. ID. NOS. 1-4). The E1A gene product has the capacity, when expressed in a malignant cell to convert the phenotype such that the resulting cell is able to inhibit the growth of neighboring tumor cells. Such capacity is herein termed E1A activity. The invention is intended to E1A, its functional subunits and fragments and modifications to the native sequence which retain E1A activity.

As used herein, the term "nucleic acid" means DNA, RNA or cDNA. A "polypeptide having E1A activity" encompasses any amino acid sequence having the biological activity of the adenovirus E1A gene product. In the preferred embodiment of this invention, this amino acid sequence is the 243 amino acid polypeptide product of the adenovirus E1A gene, as provided in FIG. 5 (SEQ. ID. NOS. 1 and 2). Functional equivalents of the 243 amino acid polypeptide are polypeptides that specifically can also effect promotion of contact inhibition as provided herein.

As used herein, the term "introducing" encompasses any method of inserting an exogenous nucleic acid molecule into a cell and includes, but is not limited to transduction, transfection, microinjection and viral infection of host cells. Methods of carrying out these procedures are well known to those of skill in the art. In the preferred embodiment of this invention, the nucleic acid is introduced into the cell by contacting a cell with a retroviral vector containing the nucleic acid under conditions such that the nucleic acid is inserted into the cell. In the most preferred embodiment of this invention, the virus is a replication-incompetent retrovirus. A replication-incompetent retrovirus is defined as a virus not having the ability to produce viral proteins, precluding spread of the vector in the infected host cells. Examples of such vectors useful for the practice of this invention are well known and readily available to those of skill in the art.

Isolated nucleic acids useful in this invention are those that encode a polypeptide functionally equivalent to a polypeptide encoded by the E1A region of the adenovirus genome. In the preferred embodiment of this invention, the isolated nucleic acid is the adenovirus E1A region. This region is defined by those of skill in the art to be from nucleotide 560 to nucleotide 1542.

This invention also provides a novel form of cancer therapy. Previously, anti-cancer genes or drugs targeted to kill tumor cells were effective in eradicating tumors only if 100% of the cells are exposed to the drug and forced to express the gene. This consistent exposure is seldom, if ever, achieved in vivo. Malignant cells transfected to express the isolated nucleic acids of this invention can dominantly prevent the growth of neighboring tumor cells. Thus, by converting a small fraction of the malignant cells, the tumor can be effectively eradicated or inhibited.

The following examples are intended to illustrate but not limit the invention.

EXAMPLE I Material and Methods

Stable E1A-expressing cell lines from A2058 melanoma and HT1080 fibrosarcoma cells (ATCC Acceession No. CCU 21) were constructed using the plasmid p1Aneo as described in Frisch et al., Oncogene 5:75-83 (1990), incorporated herein by reference. E1A-expressing HeLa cells, as described in Brunet and Berk, Mol. Cell. Biol. 8:4799-4807 (1988), incorporated herein by reference, were obtained from L. Brunet and A. Berk (University of California, Los Angeles). Cells labeled HT1080neo^(r) and A2058neo^(r) resulted from transfection with Bluescript plasmid (Stratagene, La Jolla, Calif.) containing the simian virus 40 early enhancer-promoted aph gene (encoding resistance to G418).

Cells were plated at 4×10⁴ (A2058 and derivatives), 3×10⁴ (HeLa and derivatives), or 2×10⁵ (HT1080 and derivatives) cells per 60-mm plate as described in Freedman et al., Proc. Natl. Acad. Sci. USA 72:4435-4439 (1972) and Tucker et al., Cancer Res. 37:1571-1579 (1977), which are incorporated herein by reference, and stained, after a 14- to 17-day incubation with p-iodonitrotetrazolium violet (0.5 mg/ml) for 16 h.

Cells were injected into 4- to 5-week old athymic nude mice (Harian-Sprague-Dawley) in 0.25 ml of phosphate-buffered saline at the indicated cell number. Nine mice were injected per cell line, and tumors were dissected at the indicated incubation times.

Confluent cultures of cell lines (containing 2× 10⁶ cells) were labeled for 5 hours in 35-mm wells with 0.4 mCi (1 Ci=37 GBq) of [³⁵ S] methionine (Tran³⁵ S-label, ICN) in methionine-free Dulbecco's modified Eagle's medium containing 5% (vol/vol) dialyzed fetal calf serum. Cells were washed twice in phosphate-buffered saline and scraped into 1.0 ml of RIPA-1 [50 mM Tris-HCl, pH 7.5/0.1% Nonidet P-40/250 mM NaCl/aprotinin (10 μg/ml) leupeptin (5 μg/ml) 1 mM phenylmethylsulfonyl fluoride/5 mM EDTA/soybean trypsin inhibitor (10 μg/ml)]. After addition of bovine serum albumin to 0.5 mg/ml, lysates were preabsorbed with 100 μl of a 50% (wt/vol) protein A-Sepharose (Pharmacia) slurry (prepared in RIPA-1 containing bovine serum albumin at 0.5 mg/ml) by mixing at 4° C. for 30 min and centrifuging for 10 min at 14,000 rpm in an Eppendorf microcentrifuge and 0.5-ml samples were then incubated with 1.5 μg of anti-E1A monoclonal antibody M73 (12) or control antibody [anti-fos Ab-1 (Oncogene Sciences, Mineola, N.Y.)] for 2 h at 0° C. Then 25 μl of 50% protein A-Sepharose slurry was added and the tubes were mixed for 20 min at 4° C. followed by a 2-min centrifugation and five 0.5-ml washes with RIPA-1. Pellets were then resuspended in 60 μl of sample buffer and analyzed with SDS/PAGE.

EXAMPLE II Reverse Transformation of Malignant Cells

The expression of the E1A gene was documented at the RNA level by Northern blot analysis as described in Frisch et al., Oncogene 5:75-83 (1990) and Brunet and Berk, Mol. Cell. Biol. 8:4799-4807 (1988), which are incorporated herein by reference and, in the present study, protein expression was documented by immunoprecipitation with E1A-specific monoclonal antibodies (FIG. 1). Multiple species of E1A protein were detected on the gel.

In each case, the E1A-transfected cells are relatively flat and "epithelioid" compared with the respective parental cell lines or lines transfected with the aph gene alone (FIG. 2A). These morphologic changes are consistent with the widely reported observation that transformed cells tend to be more refractile and less substrate-attached than normal cells as described in Edelman and Yahara, Proc. Natl. Acad. Sci. USA 73:2047-2051 (1976). The altered morphology was observed at the time the colonies were first identified on the G418-selection plates, suggesting that secondary stochastic changes in the genome are not required for induction of the altered phenotype.

                  TABLE 1                                                          ______________________________________                                         Tumorigenicity of parental tumor cells (HT1080, A2058, and                     HeLa) and E1A derivatives in nude mice                                                  Cells               Average                                                    injected, Incubation                                                                               tumor mass,                                       Cell line                                                                               no.       time, days                                                                               g         SD                                      ______________________________________                                         HT1080   6 × 10.sup.6                                                                       17        2.2       0.62                                    p2AHT2a  6 × 10.sup.6                                                                       17        0.04      0.04                                    HT1080   2 × 10.sup.6                                                                       17        1.2       0.3                                     p1Aneo15 2 × 10.sup.6                                                                       17        0.25      0.3                                     p1Aneo16 2 × 10.sup.6                                                                       17        0.0       0                                       A2058    1 × 10.sup.7                                                                       18        1.2       0.3                                     1A58c8-1 1 × 10.sup.7                                                                       18        0.0       0                                       1A58c11-1                                                                               1 × 10.sup.7                                                                       18        0.0       0                                       HeLa     1 × 10.sup.6                                                                       15        0.4       0.1                                     Medg 28  1 × 10.sup.6                                                                       15        0.1       0.03                                    ______________________________________                                    

The tumorigenicity of cells in vivo generally correlated with their ability to form anchorage-independent colonies (Shin et al., Proc. Natl. Acad. Sci. USA 72:4435-4439 (1972) and Tucker et al., Cancer Res. 37:1571-1579 (1977), which are hereby incorporated by reference). To test for anchorage-independent growth, cell lines were plated in soft agarose at fixed cell number and monitored for colony formation. While the parental tumor cell lines formed colonies in soft agarose (=1% efficiency), E1A-expressing clones derived from these cell lines failed to form colonies (FIG. 3). In fact, microscopic examination revealed that the E1A-expressing clones failed even to initiate colony formation on soft agarose, ruling out a growth-rate effect. The colony-suppressed phenotype was never observed among 15 independent HT1080 clones that had been transfected with the aph gene alone and selected for G418 resistance; these results indicate that the soft agarose colony suppression was an effect of the E1A gene and not of the selection process. These observations demonstrate that E1A expression can convert the human tumor cells to a phenotype that is anchorage-dependent for growth.

To test whether E1A expression rendered the human tumor cell lines non-tumorigenic in vivo, they were injected subcutaneously into nude athymic mice and monitored for tumor formation (FIG. 4). The parental cell lines formed large tumors with incubation times of 15-17 days. In contrast, the E1A-expressing A2058 and HT1080 cells produced undetectable or very small tumors. E1A only partially suppressed tumorigenicity in the HeLa cells (4-fold), possibly because transcription from the glucocorticoid-dependent murine mammary tumor virus promoter, which was driving the E1A gene, was compromised in the subcutaneous environment.

To further rule out the possibility that E1A simply slowed growth rates so as to render soft agar colonies and tumors undetectable at short time points, growth curves were constructed. E1A did not substantially affect the growth rate of subconfluent tumor cells.

The possibility that the tumor suppression could have been augmented by the ability of E1A to sensitize certain cells to lysis by tumor necrosis factor α was also considered. This was, however, unlikely in that ⁵¹ Cr-labeled p2AHT2a cells gave only 18% more lysis than parental HT1080 cells in tumor necrosis factor α at 500 units/ml--a dose high enough to kill nearly 100% of sensitive rodent cells.

This is the first time that the phenotypic effects of E1A in human cells have been described. The experiments reported here establish that E1A converts three diverse types of human tumor cells into a phenotypically non-transformed state. Paradoxically, a salient phenotypic effect of adenovirus E1A is collaboration with the ras oncogene to transform primary rodent cells. Since specific rodent cell types can turn out to be affected antioncogenically by E1A, it would be premature to ascribe this discrepancy to species differences only, especially since the effects of E1A expression in spontaneously transformed rodent cell lines have not been reported.

EXAMPLE III Inhibition of Tumor Cell Growth By Contact Inhibition

This example describes a method for inhibiting the uncontrolled replication of human tumor cells through contact inhibition with cells reverse transformed by E1A expression.

Construction of E1A viral expression vectors was achieved by subcloning cDNAs encoding the 243 amino acid (SEQ. ID. NOS. 1 and 2) or 289 amino acid (SEQ. ID. NOS. 3 and 4) E1A proteins of adenovirus type 5 into the retroviral vectors: LNCX, LNSX. (For vector descriptions, see Miller and Rosman, Biotechniques 7:980-990 (1989); details of plasmid constructions are given in the legend to FIG. 6). 10 μg of these plasmid DNAs were transfected as in Example II into the ecotropic packaging cell line gpE86 (maintained in hygromycin/mycophenolic acid selection medium). At 48 h post transfection, unselected virus stocks were prepared by collecting conditioned media, clearing by centrifugation and freezing. These viral stocks were used to infect the amphotropic packaging cell line gpEam12 (cells available from ATCC) in 35 mm wells by incubation for 12 h in DME/10% fetal calf serum media containing 4 μg/ml polybrene. At 24 h post-infection, cells were split at ratios between 1:100-1:300, and infected cells were selected in 500 μg/ml G418 for 3 weeks. Virus-producing cell lines were expanded into 35 mm wells and conditioned media containing virus was prepared for each line. Virus stocks were prepared from producer cell lines containing either 243 amino acid E1A or 289 amino acid E1A, in the vectors LNCX and LNSX (which promote transcription using internal CMV or SV40 early enhancers, respectively).

These stocks were used to infect the human fibrosarcoma line HT1080 by incubating 0.4 ml of virus stock with a 35 mm well of HT1080 cells for 8 h in medium containing polybrene; infected cells were split at various ratios 24 h post-infection, and either G418-resistant clones or mixed populations of infected cells were analyzed further.

The results were as follows. In cultures infected with some of the virus stocks, no morphologic change was observed compared with the uninfected controls. Western blotting demonstrated that these infected cultures were negative for E1A expression, possibly as a result of rearrangement of retroviral DNA sequences in the producer cell lines.

With one of the virus stocks, encoding the 243 amino acid form of E1A in the LNSX vector (especially 4/LNSX8), essentially 100% of the cells were converted into a morphology closely resembling that of E1A-transfected cells (see FIG. 2). Upon prolonged incubation, these cells maintained their epithelioid morphology and failed to grow beyond monolayer stage, suggesting contact inhibition of growth. These cells expressed an amount of E1A protein per cell comparable with the levels seen in calcium phosphate-transfected cell lines, but all of this E1A was the 243 amino acid form, as expected.

With other virus stocks, a mixture of "transformed morphology" and "E1A morphology" clones was noted after G418 selection. Significantly, upon longer incubation, these cultures were found to eventually (2-3 weeks post-infection) convert into nearly 100% "E1A morphology"; i.e., ephithelioid, contact-inhibited cells.

The latter phenomenon indicates that cells expressing E1A contact-inhibit each other. They also contact-inhibit tumor cells. Consistant with this observation is that the tumor cells are incapable of "sending" contact inhibition signals as expected, but are capable of "receiving" and responding to them. These differential signaling responses eventually produce the outcome described in the preceding paragraph. Thus, contact inhibition can be achieved within an hyperproliferating cell population by expressing E1A in a fraction of the cells and not within the total population.

The conceptual advance bearing important ramifications for cancer therapy is as follows. Genes or drugs targeted to kill tumor cells are effective in eradicating tumors only if 100% of the cells are exposed to the drug or forced to express the gene. This condition is seldom, if ever, satisfied in vivo. E1A-expressing cells can dominantly prevent the growth of neighboring tumor cells, so forcing a small fraction of the cells in a tumor to express E1A will to eradicate the tumor. Delivery of E1A by retroviral vectors will achieve this effect. E1A is unique among tumor suppressor genes presently characterized in preserving the viability of the cells into which it is introduced, and this property can be exploited to eradicate tumor growth.

Although the invention has been described with reference to the disclosed embodiments, those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention. It should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 4                                                   (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1000 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: both                                                         (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (iii) HYPOTHETICAL: NO                                                          (iv) ANTI-SENSE: NO                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: join(10..423, 678..993)                                          (D) OTHER INFORMATION: /note="This protein is the product                      of alternative splicing of the precursor RNA such                              that protein in this form is a subset of the                                   larger E1A protein as shown in SEQ ID NOS. 3-4."                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        GGACTGAAAA TGAGACATATTATCTGCCACGGAGGTGTTATTACCGAA48                            MetArgHisIleIleCysHisGlyGlyValIleThrGlu                                        1510                                                                           GAAATGGCCGCCAGT CTTTTGGACCAGCTGATCGAAGAGGTACTGGCT96                            GluMetAlaAlaSerLeuLeuAspGlnLeuIleGluGluValLeuAla                               152025                                                                         GATAATCTTCCACCTCCTAGC CATTTTGAACCACCTACCCTTCACGAA144                           AspAsnLeuProProProSerHisPheGluProProThrLeuHisGlu                               30354045                                                                       CTGTATGATTTAGAC GTGACGGCCCCCGAAGATCCCAACGAGGAGGCG192                           LeuTyrAspLeuAspValThrAlaProGluAspProAsnGluGluAla                               505560                                                                         GTTTCGCAGATT TTTCCCGACTCTGTAATGTTGGCGGTGCAGGAAGGG240                           ValSerGlnIlePheProAspSerValMetLeuAlaValGlnGluGly                               657075                                                                         ATTGACTTACTC ACTTTTCCGCCGGCGCCCGGTTCTCCGGAGCCGCCT288                           IleAspLeuLeuThrPheProProAlaProGlySerProGluProPro                               808590                                                                         CACCTTTCCCGGCAG CCCGAGCAGCCGGAGCAGAGAGCCTTGGGTCCG336                           HisLeuSerArgGlnProGluGlnProGluGlnArgAlaLeuGlyPro                               95100105                                                                       GTTTCTATGCCAAACCTTGTA CCGGAGGTGATCGATCTTACCTGCCAC384                           ValSerMetProAsnLeuValProGluValIleAspLeuThrCysHis                               110115120125                                                                   GAGGCTGGCTTTCCA CCCAGTGACGACGAGGATGAAGAGGGTGAGGAGT433                          GluAlaGlyPheProProSerAspAspGluAspGluGlu                                        130135                                                                         TTGTGTTAGATTATGTGGAGCACCCCGGGCACGGTTGCAGGTC TTGTCATTATCACCGGA493               GGAATACGGGGGACCCAGATATTATGTGTTCGCTTTGCTATATGAGGACCTGTGGCATGT553                TTGTCTACAGTAAGTGAAAATTATGGGCAGTGGGTGATAGAGTGGTGGGTTTGGTGTGGT613                AATTTTTTTTTTAATTTT TACAGTTTTGTGGTTTAAAGAATTTTGTATTGTGATTTTTTT673               AAAAGGTCCTGTGTCTGAACCTGAGCCTGAGCCCGAGCCAGAACCGGAG722                           GlyProValSerGluProGluProGluProGluProGluProGlu                                   140145150                                                                     CCTGCAAGACCTACCCGCCGTCCTAAAATGGCGCCTGCTATCCTGAGA770                            ProAlaArgProThrArgArgProLysMetAlaProAlaIleLeuArg                                155160165                                                                     CGCCCGACATCACCTGTGTCTAGAGAATGCAATAGTAGTACGGATAGC818                            ArgProThrSerProValSerArgGluCysAsnSerSerThrAspSer                               170 175180185                                                                  TGTGACTCCGGTCCTTCTAACACACCTCCTGAGATACACCCGGTGGTC866                            CysAspSerGlyProSerAsnThrProProGluIleHisProValVal                                190195200                                                                     CCGCTGTGCCCCATTAAACCAGTTGCCGTGAGAGTTGGTGGGCGTCGC914                            ProLeuCysProIleLysProValAlaValArgValGlyGlyArgArg                                205210215                                                                     CAGGCTGTGGAATGTATCGAGGACTTGCTTAACGAGCCTGGGCAACCT962                            GlnAlaValGluCysIleGluAspLeuLeuAsnGluProGlyGlnPro                                220225230                                                                     TTGGACTTGAGCTGTAAACGCCCCAGGCCATAAGGTGT1000                                     LeuAspLeuSerCysLysArgProArgPro                                                 235240                                                                         (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 243 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        MetArgHisIleIleCysHisGlyGlyValIleThrGluGluMetAla                               1 51015                                                                        AlaSerLeuLeuAspGlnLeuIleGluGluValLeuAlaAspAsnLeu                               202530                                                                         ProProProSerHisPheGlu ProProThrLeuHisGluLeuTyrAsp                              354045                                                                         LeuAspValThrAlaProGluAspProAsnGluGluAlaValSerGln                               5055 60                                                                        IlePheProAspSerValMetLeuAlaValGlnGluGlyIleAspLeu                               65707580                                                                       LeuThrPheProProAlaProGlySerProGluProProHis LeuSer                              859095                                                                         ArgGlnProGluGlnProGluGlnArgAlaLeuGlyProValSerMet                               100105110                                                                      Pr oAsnLeuValProGluValIleAspLeuThrCysHisGluAlaGly                              115120125                                                                      PheProProSerAspAspGluAspGluGluGlyProValSerGluPro                               130 135140                                                                     GluProGluProGluProGluProGluProAlaArgProThrArgArg                               145150155160                                                                   ProLysMetAlaProAlaIleLeu ArgArgProThrSerProValSer                              165170175                                                                      ArgGluCysAsnSerSerThrAspSerCysAspSerGlyProSerAsn                               180185 190                                                                     ThrProProGluIleHisProValValProLeuCysProIleLysPro                               195200205                                                                      ValAlaValArgValGlyGlyArgArgGlnAlaValGluCys IleGlu                              210215220                                                                      AspLeuLeuAsnGluProGlyGlnProLeuAspLeuSerCysLysArg                               225230235240                                                                   ProAr gPro                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1000 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: both                                                         (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: join(10..561, 678..993)                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                        GGACTGAAAATGAGACATATTATCTGCCACGGAGGTGTTATTACCGAA48                             MetArgHisIleIleCysHisGlyGlyValIleThrGlu                                        15 10                                                                          GAAATGGCCGCCAGTCTTTTGGACCAGCTGATCGAAGAGGTACTGGCT96                             GluMetAlaAlaSerLeuLeuAspGlnLeuIleGluGluValLeuAla                               15202 5                                                                        GATAATCTTCCACCTCCTAGCCATTTTGAACCACCTACCCTTCACGAA144                            AspAsnLeuProProProSerHisPheGluProProThrLeuHisGlu                               303540 45                                                                      CTGTATGATTTAGACGTGACGGCCCCCGAAGATCCCAACGAGGAGGCG192                            LeuTyrAspLeuAspValThrAlaProGluAspProAsnGluGluAla                               5055 60                                                                        GTTTCGCAGATTTTTCCCGACTCTGTAATGTTGGCGGTGCAGGAAGGG240                            ValSerGlnIlePheProAspSerValMetLeuAlaValGlnGluGly                               6570 75                                                                        ATTGACTTACTCACTTTTCCGCCGGCGCCCGGTTCTCCGGAGCCGCCT288                            IleAspLeuLeuThrPheProProAlaProGlySerProGluProPro                               8085 90                                                                        CACCTTTCCCGGCAGCCCGAGCAGCCGGAGCAGAGAGCCTTGGGTCCG336                            HisLeuSerArgGlnProGluGlnProGluGlnArgAlaLeuGlyPro                               9510010 5                                                                      GTTTCTATGCCAAACCTTGTACCGGAGGTGATCGATCTTACCTGCCAC384                            ValSerMetProAsnLeuValProGluValIleAspLeuThrCysHis                               110115120 125                                                                  GAGGCTGGCTTTCCACCCAGTGACGACGAGGATGAAGAGGGTGAGGAG432                            GluAlaGlyPheProProSerAspAspGluAspGluGluGlyGluGlu                               130135 140                                                                     TTTGTGTTAGATTATGTGGAGCACCCCGGGCACGGTTGCAGGTCTTGT480                            PheValLeuAspTyrValGluHisProGlyHisGlyCysArgSerCys                               145150 155                                                                     CATTATCACCGGAGGAATACGGGGGACCCAGATATTATGTGTTCGCTT528                            HisTyrHisArgArgAsnThrGlyAspProAspIleMetCysSerLeu                               160165 170                                                                     TGCTATATGAGGACCTGTGGCATGTTTGTCTACAGTAAGTGAAAATTATGGGC581                       CysTyrMetArgThrCysGlyMetPheValTyr                                              175180                                                                         AGTGGGTGATAGAGTGGTGGGTTTGGTGTGGT AATTTTTTTTTTAATTTTTACAGTTTTG641               TGGTTTAAAGAATTTTGTATTGTGATTTTTTTAAAAGGTCCTGTGTCTGAACCT695                      GlyProValSerGluPro                                                              185190                                                                        GAGCCTGAGCCCGAGCCAGAACCGGAGCCTGCAAGACCTACCCGCCGT743                            GluProGluProGluProGluProGluProAlaArgProThrArgArg                                195200205                                                                     CCTAAAATGGCGCCTGCTATCCTGAGACGCCCGACATCACCTGTGTCT791                            ProLysMetAlaProAlaIleLeuArgArgProThrSerProValSer                                210215220                                                                     AGAGAATGCAATAGTAGTACGGATAGCTGTGACTCCGGTCCTTCTAAC839                            ArgGluCysAsnSerSerThrAspSerCysAspSerGlyProSerAsn                                225230235                                                                     ACACCTCCTGAGATACACCCGGTGGTCCCGCTGTGCCCCATTAAACCA887                            ThrProProGluIleHisProValValProLeuCysProIleLysPro                               240 245250                                                                     GTTGCCGTGAGAGTTGGTGGGCGTCGCCAGGCTGTGGAATGTATCGAG935                            ValAlaValArgValGlyGlyArgArgGlnAlaValGluCysIleGlu                               255 260265270                                                                  GACTTGCTTAACGAGCCTGGGCAACCTTTGGACTTGAGCTGTAAACGC983                            AspLeuLeuAsnGluProGlyGlnProLeuAspLeuSerCysLysArg                                275280285                                                                     CCCAGGCCATAAGGTGT1000                                                          ProArgPro                                                                      (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 289 amino acids                                                    ( B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                        MetArgHisIleIleCysHisGlyGlyValIleThrGluGluMetAla                               151015                                                                         AlaSerLeu LeuAspGlnLeuIleGluGluValLeuAlaAspAsnLeu                              202530                                                                         ProProProSerHisPheGluProProThrLeuHisGluLeuTyrAsp                               35 4045                                                                        LeuAspValThrAlaProGluAspProAsnGluGluAlaValSerGln                               505560                                                                         IlePheProAspSerValMetLeuAlaValGlnGluG lyIleAspLeu                              65707580                                                                       LeuThrPheProProAlaProGlySerProGluProProHisLeuSer                               8590 95                                                                        ArgGlnProGluGlnProGluGlnArgAlaLeuGlyProValSerMet                               100105110                                                                      ProAsnLeuValProGluValIleAspLeuThrCysHisGluAlaGl y                              115120125                                                                      PheProProSerAspAspGluAspGluGluGlyGluGluPheValLeu                               130135140                                                                      AspTyrValGluHisPro GlyHisGlyCysArgSerCysHisTyrHis                              145150155160                                                                   ArgArgAsnThrGlyAspProAspIleMetCysSerLeuCysTyrMet                               165 170175                                                                     ArgThrCysGlyMetPheValTyrGlyProValSerGluProGluPro                               180185190                                                                      GluProGluProGluProGluProAlaA rgProThrArgArgProLys                              195200205                                                                      MetAlaProAlaIleLeuArgArgProThrSerProValSerArgGlu                               210215220                                                                       CysAsnSerSerThrAspSerCysAspSerGlyProSerAsnThrPro                              225230235240                                                                   ProGluIleHisProValValProLeuCysProIleLysProValAla                                245250255                                                                     ValArgValGlyGlyArgArgGlnAlaValGluCysIleGluAspLeu                               260265270                                                                      LeuAsnGlu ProGlyGlnProLeuAspLeuSerCysLysArgProArg                              275280285                                                                      Pro                                                                        

I claim:
 1. A method for promoting the differentaiation of a pathologically hyperproliferative human cell, wherein said pathologically hyperproliferative cell does not overexpress the neu oncogene and further wherein the pathologically hyerproliferative human cell is selected from the group consisting of sarcoma, carcinoma, and melanoma, comprising contacting said pathologically hyperproliferative cell in vitro with nucleic acid encoding a polyeptide having E1A activity under conditions such that said nucleic acid is introduced and said polpeptide is expressed in said pathologically hyperproliferative cell.
 2. The method of claim 1, wherein the isolated nucleic acid encodes adenovirus E1A polypeptide.
 3. The method of claim 1, wherein the pathologic hyperproliferative cell is a malignant cell.
 4. The method of claim 1, wherein the isolated nucleic acid is introduced into the cell by contacting the cell with a suitable retroviral vector containing the isolated nucleic acid sequence.
 5. A method for reverse-transforming a pathologically hyperproliferative human cell, wherein said pathologically hyperproliferative cell does not overexpress the neu oncogene and further wherein the pathologically hyperproliferative human cell is selected from the group consisting of sarcoma, carcinoma, and melanoma, comprising contacting said hyperproliferative cell in vitro with nucleic acid encoding a polypeptide having E1A activity under conditions such that said nucleic acid is introduced and said polypeptide is expressed in said pathologically hyperproliferative cell.
 6. The method of claim 5, wherein the isolated nucleic acid encodes adenovirus E1A polypeptide.
 7. The method of claim 5, wherein the pathologic hyperproliferative cell is a malignant cell.
 8. The method of claim 5, wherein the isolated nucleic acid is introduced into the cell by contacting the cell with a suitable retroviral vector containing the isolated nucleic acid sequence.
 9. The method of claim 1, wherein the sarcoma is selected from the group consisting of fibrosarcoma, osteosarcoma, rhabdomyosarcoma, and breast cancer.
 10. The method of claim 1, wherein the sarcoma is selected from the group consisting of fibrosarcoma, osteosarcoma, rhabdomyosarcoma, and breast cancer.
 11. The method of claim 1, wherein the polypeptide having E1A activity is the 243 amino acid polypeptide product of the adenovirus E1A gene as shown in FIG. 5 (SEQ ID NO. 2).
 12. The method of claim 1, wherein the polypeptide having E1A activity is the 243 amino acid polypeptide product of the adenovirus E1A gene as shown in FIG. 5 (SEQ ID NO. 2). 